The broad objective of this proposal is to understand the molecular mechanisms that regulate the activation status and function of low versus high avidity tumor-antigen specific T cells in non-tolerized and tolerized mice. Identification of these mechanisms is the first step toward developing approaches that can allow for more effective activation of T cells with a range of avidities specific for cancer antigens. The success of immunization against many viruses is in part due to the induction of a virus specific, high avidity CD8+ T cell repertoire with a sufficient memory T cell compartment in healthy hosts. In contrast, successful vaccination in cancer bearing hosts requires bypassing multiple mechanisms of immune tolerance. Many of the known tumor antigens are over-expressed tissue- specific antigens. Mechanisms of T cell tolerance must exist to prevent autoimmunity against self- antigens. These same mechanisms that establish and maintain self-tolerance are likely contributors to the lack of effectiveness of T cells in vivo that is often observed in cancer patients. The implications of tolerance induction are that cancer-specific high avidity T cell populations are deleted or suppressed, and that cancer vaccines must either break tolerance in anergic T cells or activate populations of low avidity T cells which, based on their weak reactivity, may escape active tolerance induction. Until recently, the tools to understand the fate of the high avidity and low avidity cancer specific CD8+ T cell repertoires in cancer bearing hosts were not available. The HER-2/neu transgenic (neu-N) mouse model of spontaneous mammary tumors provides the opportunity to evaluate this issue using a natural tumor antigen, HER-2/neu (neu). In vaccinated parental FVB/N mice, the majority of CD8+ T cells are of high avidity and directed against an immunodominant antigen, RNEU420-429. In contrast, in vaccinated neu-N mice, most T cell responses are weak, of low avidity, and directed against multiple epitopes. To understand the mechanisms that regulate the activation and function of CD8+ T cells of both low and high avidity, we have developed high and low avidity RNEU420-429-specific TCR transgenic mice. In aim 1 we will compare and characterize the fate of the high avidity versus the low avidity RNEU420-429-specific CD8+ T cells in vivo, and determine the role regulatory T cells (Tregs) play in controlling the function of activated and memory high avidity versus low avidity T cells in vivo. In aim 2, we will evaluate the role of microRNA (miRNA) as post- transcriptional regulators of T cell function in the high avidity versus low avidity RNEU420-429-specific CD8+ T cells. In aim 3, we will evaluate the high avidity versus low avidity RNEU420-429-specific CD8+ TCR transgenic T cells for changes in T cell signaling pathways. PUBLIC HEALTH RELEVANCE: The broad objective of this proposal is to understand the molecular mechanisms that regulate the activation status and function of low versus high avidity tumor-antigen specific T cells in non-tolerized and tolerized mice. Specifically, we will evaluate a number of mechanisms that may differentially regulate low versus high avidity T cells including: regulatory T cells, T cell signaling pathways, and post-transcriptional regulation.